Molecular, Cellular and Physiological Evidences for the Anorexigenic Actions of Nesfatin-1 in Goldfish

Nesfatin-1 is a recently discovered anorexigen encoded in the precursor peptide, nucleobindin-2 (NUCB2) in mammals. To date, nesfatin-1 has not been described in any non-mammalian species, although some information is available in the sequenced genomes of several species. Our objective was to characterize nesfatin-1 in fish.In the present study, we employed molecular, immunohistochemical, and physiological studies to characterize the structure, distribution, and appetite regulatory effects of nesfatin-1 in a non-mammalian vertebrate. A very high conservation in NUCB2 sequences, especially in the nesfatin-1 region was found in lower vertebrates. Abundant expression of NUCB2 mRNA was detected in several tissues including the brain and liver of goldfish. Nesfatin-1-like immunoreactive cells are present in the feeding regulatory nucleus of the hypothalamus and in the gastrointestinal tract of goldfish. Approximately 6-fold increase in NUCB2 mRNA levels was found in the liver after 7-day food-deprivation, and a similar increase was also found after short-term fasting. This points toward a possible liver specific role for NUCB2 in the control of metabolism during food-deprivation. Meanwhile, ∼2-fold increase at 1 and 3 h post-feeding and an ∼3-fold reduction after a 7-day food-deprivation was observed in NUCB2 mRNA in the goldfish hypothalamus. In vivo, a single intraperitoneal injection of the full-length native (goldfish; gf) nesfatin-1 at a dose of 50 ng/g body weight induced a 23% reduction of food intake one hour post-injection in goldfish. Furthermore, intracerebroventricular injection of gfnesfatin-1 at a dose of 5 ng/g body weight resulted in ∼50% reduction in food intake.Our results provide molecular, anatomical and functional evidences to support potential anorectic and metabolic roles for endogenous nesfatin-1 in goldfish. Collectively, we provide novel information on NUCB2 in non-mammals and an anorexigenic role for nesfatin-1 in goldfish

Xenin is a novel anorexigen in goldfish (Carassius auratus).

Xenin, a highly conserved 25 amino acid peptide cleaved from the N-terminus of the coatomer protein alpha (COPA), is emerging as a food intake regulator in mammals and birds. To date, no research has been conducted on xenin biology in fish. This study aims to identify the copa mRNA encoding xenin in goldfish (Carassius auratus) as a model, to elucidate its regulation by feeding, and to describe the role of xenin on appetite. First, a partial sequence of copa cDNA, a region encoding xenin, was identified from goldfish brain. This sequence is highly conserved among both vertebrates and invertebrates. RT-qPCR revealed that copa mRNAs are widely distributed in goldfish tissues, with the highest levels detected in the brain, gill, pituitary and J-loop. Immunohistochemistry confirmed also the presence of COPA peptide in the hypothalamus and enteroendocrine cells on the J-loop mucosa. In line with its anorexigenic effects, we found important periprandial fluctuations in copa mRNA expression in the hypothalamus, which were mainly characterized by a gradually decrease in copa mRNA levels as the feeding time was approached, and a gradual increase after feeding. Additionally, fasting differently modulated the expression of copa mRNA in a tissue-dependent manner. Peripheral and central injections of xenin reduce food intake in goldfish. This research provides the first report of xenin in fish, and shows that this peptide is a novel anorexigen in goldfish

Effects of IP (A) and ICV (B) administration of xenin on food intake.

<p>Data are presented as mean + SEM (n = 6 fish for IP, 4 fish for ICV). Columns with a different letter are statistically different (p < 0.05, one-way ANOVA and Student-Newman-Keuls tests).</p

Xenin is a novel anorexigen in goldfish (<i>Carassius auratus</i>)

<div><p>Xenin, a highly conserved 25 amino acid peptide cleaved from the N-terminus of the coatomer protein alpha (COPA), is emerging as a food intake regulator in mammals and birds. To date, no research has been conducted on xenin biology in fish. This study aims to identify the <i>copa</i> mRNA encoding xenin in goldfish (<i>Carassius auratus</i>) as a model, to elucidate its regulation by feeding, and to describe the role of xenin on appetite. First, a partial sequence of <i>copa</i> cDNA, a region encoding xenin, was identified from goldfish brain. This sequence is highly conserved among both vertebrates and invertebrates. RT-qPCR revealed that <i>copa</i> mRNAs are widely distributed in goldfish tissues, with the highest levels detected in the brain, gill, pituitary and J-loop. Immunohistochemistry confirmed also the presence of COPA peptide in the hypothalamus and enteroendocrine cells on the J-loop mucosa. In line with its anorexigenic effects, we found important periprandial fluctuations in <i>copa</i> mRNA expression in the hypothalamus, which were mainly characterized by a gradually decrease in <i>copa</i> mRNA levels as the feeding time was approached, and a gradual increase after feeding. Additionally, fasting differently modulated the expression of <i>copa</i> mRNA in a tissue-dependent manner. Peripheral and central injections of xenin reduce food intake in goldfish. This research provides the first report of xenin in fish, and shows that this peptide is a novel anorexigen in goldfish.</p></div

Pre- and post-prandial changes of <i>copa</i> mRNA expression in the goldfish hypothalamus (A), J-loop (B), and liver (C).

<p>The mRNA expression of <i>copa</i> was normalized to <i>β-actin</i> and represented relative to the -3 h scheduled feeding group. Data are presented as mean + SEM (n = 4 fish). Columns sharing a same letter are not statistically different (p < 0.05, one-way ANOVA and Student-Newman-Keuls tests).</p

Fasting-induced changes of <i>copa</i> mRNA expression in goldfish central and peripheral tissues.

<p>(<b>A–C</b>) Expression of <i>copa</i> mRNAs after a 3-day fasting period in the goldfish hypothalamus (A), J-loop (C), and liver (E). The mRNA expression of <i>copa</i> was normalized to <i>β-actin</i> and represented relative to the unfed group. Data are presented as mean + SEM (n = 4 fish). Columns with a different letter are statistically different (p < 0.05, t-test). (<b>D–F</b>) Expression of <i>copa</i> mRNAs after a 7-day fasting period and refeeding in the goldfish hypothalamus (D), J-loop (E), and liver (F). The mRNA expression of <i>copa</i> was normalized to <i>β-actin</i> and represented relative to the unfed group. Data are presented as mean + SEM (n = 4 fish). Columns with a different letter are statistically different (p < 0.05, one-way ANOVA and Student-Newman-Keuls tests).</p

Phylogenetic analysis of the partial COPA sequence obtained from goldfish.

<p><b>(A)</b> Phylogenetic tree showing the evolutionary relationships of the obtained nucleotide sequence of goldfish <i>copa</i> with those of other species. Tree was inferred by the neighbor-joining method using the online tool <a href="http://www.phylogeny.fr" target="_blank">www.phylogeny.fr</a>. The scale bar indicates the average number of substitutions per position (a relative measure of evolutionary distance). The names of the species used for the alignment are provided in the figure. GenBank accession numbers of the sequences used are as follows: <i>Bombyx mori</i>, NM_001172721.1; <i>Bos taurus</i>, NM_001105645.1; <i>Carassius auratus</i>, JQ929912.1; <i>Cavia porcellus</i>, XM_003466569.3; <i>Ciona intestinalis</i>, XM_002131228.4; <i>Cricetulus griseus</i>, XM_007629307.2; <i>Danio rerio</i>, NM_001001941.2; <i>Equus caballus</i>, XM_023640888.1; <i>Gallus gallus</i>, NM_001031405.2; <i>Homo sapiens</i>, NM_001098398.1; <i>Loxodonta africana</i>, XM_023554162.1; <i>Macaca mulatta</i>, XM_015113467.1; <i>Meleagris gallopavo</i>, XM_003213951.3; <i>Monodelphis domestica</i>, XM_016430274.1; <i>Mus musculus</i>, NM_009938.4; <i>Nomascus leucogenys</i>, XM_012510889.1; <i>Pan troglodytes</i>, XM_001171563.4; <i>Rattus norvegicus</i>, NM_001134540.1; <i>Saccoglossus kowalevskii</i>, XM_002731243.2; <i>Salmo salar</i>, XM_014140665.1; <i>Sus scrofa</i>, XM_001928697.6; <i>Thalassiosira pseudonana</i>, XM_002291058.1; <i>Xenopus laevis</i>, NM_001093019.2; <i>Xenopus tropicalis</i>, NM_001127994.1. <b>(B)</b> Alignment of the first 61 aa of COPA from an algal species, invertebrate species, teleost fishes, amphibians, avians, and mammals. The species names are provided on the left-hand side of the alignment and the number of aa is present on the right-hand side of the alignment. The coloured aa highlights the differences in conservation between species. The first 25 aa (which correspond to the xenin region) are boxed.</p

Xenin-like immunoreactivity in the goldfish brain.

<p>(<b>A</b>) Sagittal view of a goldfish brain stained with DAPI (blue). Arrows indicate the region of the posterior periventricular nucleus (NPP<i>v</i>), nucleus anterior tuberis (NAT), and posterior nucleus lateralis tuberis (NLT<i>p</i>). Scale bar = 500 μm. (<b>B–D</b>) Immunohistochemical staining of goldfish NAT, NLT<i>p</i> and NPP<i>v</i> neurons for xenin-like immunoreactivity (red). All images are merged with DAPI showing nuclei in blue. Arrows point to immunopositive cells. In C, open arrows point to xenin-like immunoreactive neurons along the ventricle, and closed arrows to positive neurons lateral to the ventricle. Scale bars = 50 μm (B), 100 μm (C, D), 20 μm (inset in D). (<b>E</b>) Image of a negative control slide stained with secondary antibody alone. Scale bar = 50 μm. (<b>F, G</b>) Transversal representative sections of goldfish brain treated with specific primary anti-xenin antibody pre-absorbed in xenin. Scale bars = 200 μm.</p

Differential expression of <i>copa</i> mRNA in goldfish tissues.

<p>Real-time quantitative PCR (RT-qPCR) was used to quantify the level of <i>copa</i> mRNA expression in each tissue. The mRNA expression detected was normalized to <i>β-actin</i> and represented relative to the brain (tissue with the highest expression). Data are presented as mean + SEM (n = 6 fish). Agarose gel image of the PCR products is shown at the top.</p

Sequences of forward and reverse primers used in this study.

<p>Sequences of forward and reverse primers used in this study.</p